Almost all the skin and mucosa injuries, whether of bacterial, viral, immunological or traumatic origin, involve cellular damage, and their healing is directly related to the replacement of damaged cells. In order to grow, cells need a hydrated and clean, pathogen-free, chemical-free, waste protein- and proteolytic enzyme-free environment. Under these conditions, the mother cells secrete a cellular matrix composed of free particles of collagen, elastin, laminin, fibronectin and other proteins, which form a cushion for the attachment of daughter cells. In absence of this matrix, daughter cells have no support to attach onto and therefore cannot grow; consequently, the injury cannot heal.
Topical skin and mucosa injuries involving cellular damage are extremely difficult to heal as, in addition to containing dead cells, cell debris, and multiple proteolytic enzymes which impede cell growth and wound repair, those lesions remain open to the external environment and often get contaminated. This is the reason why most chronic wounds such as bedsores, diabetic ulcers, or venous leg ulcers, never heal, and nearly 40% patients die before they experience wound resolution.
Currently, there is no single substance or product having all the multiple and essential properties needed, and there is no complete treatment available for skin and mucosa injuries. For example, there is presently no treatment for healing chronic wounds. All the drugs available either act as antiseptics, antibiotics to reduce microbial growth, as hydrogels or alginates to keep the wound hydrated, as analgesics or anesthetics to reduce the pain, as collagen- or hyaluronic acid-containing preparations to supply one of the cellular matrix' components, or consist of bioengineered skin or epidermal grafts to replace the skin, but none of these treatments is directed to clean the injury and to favor the natural skin or mucosa repair process by promoting cell growth.
The same lack of efficient treatment is true for topical viral infections such as labial herpes, genital herpes, rhinosinusitis, and influenza, where huge amounts of free virus particles are present on the infected biological surface and continue attacking new healthy cells with the help of topically available proteases. A virus infection is often mediated through multiple proteases present on the surface of the injury. Presently, there is no drug to reduce the amount of free virus particles or of proteases, the main agents of infection. To treat viral throat infection, sea water or salt water gargarism is still considered one of the best remedies as these osmotically active solutions form a hypertonic film over the throat mucosa and the resulting outward exudation of hypotonic liquid helps reduce the contaminant load on the throat surface. Unfortunately, this hypertonic solution film gets diluted within a few minutes by the outflowing hypotonic liquid, limiting the efficacy of that treatment. Increasing the concentration of salt or other osmotic ingredients is not recommended because of the resulting strong irritation, cellular damage and mucosa burning, or because the presence of a toxic chemical in the vicinity of cells would block cell growth.
Therefore, it is highly important to find a substance capable of fulfilling the multiple requirements of cleaning the infected surface of all the contaminants, such as free-floating bacteria, virus particles, dead cells, cell debris, dead proteins, matrix metalloproteases, growth factors, cytokines, proteases, or dust particles, so as to provide the environment essential for cell growth and tissue repair, without any toxic effect on the cells or the cellular matrix.
Glycerol, a polyol with multiple hydroxyl groups, is a viscous, transparent, safe, osmotically active, hydrophilic solution of natural or synthetic origin. Glycerol is commonly used as a food ingredient, preservative, and humectant, and enters into the composition of many cosmetics and pharmaceuticals. Glycerol is widely employed for topical application on wounds and burns, often as an excipient in association with other active ingredients. In all these preparations, glycerol is used as vehicle, diluent, viscosity enhancer, humidifier, or taste modulator but not as an active ingredient to treat a topical skin or mucosa pathology.
An injured skin or mucosa acts as a semi-permeable membrane and allows osmosis to occur. The flow of solvents across a semi-permeable membrane constitutes the osmotic flow or osmosis. The pressure required to achieve osmotic equilibrium is known as osmotic pressure. The higher the concentration of a solvent, the more osmotic pressure it exerts. Fresh water is considered isotonic with a density of 1000 kg/m3. Seawater and glycerol have densities of 1025 kg/m3 and 1259 kg/m3, respectively. Osmotic pressure exerted by glycerol is nearly 10 times higher compared to seawater. The solute concentration of glycerol is also much higher compared to seawater. It is the total solute concentration in a solution that determines the osmotic flow of liquid, as osmosis is driven by differences in solute/solvent concentrations that exist across a semi-permeable membrane. The total solute concentration can be determined through the osmolality of the solution. The osmolality of seawater, containing 3.4% NaCl, is 0.581 mols/kg (total solute/mol wt/kg), compared to 10.86 mols/kg for glycerol. This shows that pure glycerol is nearly 18 times more osmotically active than seawater. Therefore, glycerol could have been a product of choice for application on infected surfaces in order to generate osmotic outflow of hypotonic liquid and to clean the injury. Unfortunately, the exudating hypotonic liquid immediately dilutes the glycerol and drains it from the wound surface within a few minutes, limiting these cleaning effects.
There is therefore a need for a safe and non-irritant treatment with the multiple properties of being hydrating, cleaning, antiseptic, and also having antibacterial, antiviral, and anti-protease activity.
WO 00/74668 discloses the use of hypertonic osmotically active solutions for the treatment of topical injuries concerning the use of glycerol as viscous solution for topical application for the treatment of ulcers or superficial injuries, comprising glycerol as active principle and a hydro-glycerinated extract of Alchemilla vulgaris as cell growth-promoting agent. However, the glycerol used in WO 00/74668 is not filmogen and therefore cannot provide for the above detailed advantages. The use of glycerol alone as a therapeutic agent to clean infected or damaged tissue is not common because, due to osmosis, the phenomenon of hypotonic liquid outflow from the inner parts of the tissue, glycerol gets diluted within a few minutes, progressively loses its osmotic properties and activity, and does not satisfactorily act as a total cleaning agent. The glycerol film should remain on the semi-permeable live tissue membrane for at least 30 minutes to generate sufficient osmosis to clean the biological tissue surface.
The present invention deals with the problem of how to improve the retention time of the glycerol film by a live biological surface in order to overcome the above-mentioned disadvantages.
Secondly, Cryopreservation is a process where cells, whole tissues, or any other substances susceptible to sustain damage caused by chemical reactivity or time, are preserved by cooling to subzero temperatures. At low enough temperatures, any enzymatic or chemical activity, which might cause damage to the material in question, is effectively stopped. Cryopreservation methods seek to reach low temperatures without engendering additional damage caused by the formation of ice crystals during freezing. For long term live cell storage, the cryoprotectant must be non-toxic to the cells and be able to prevent crystal formation. This is particularly important for live cell and tissue storage or preservation (example: skin graft) since the chances of successful cell revival or tissue grafting diminish as the number of dead or dying cells increases.
Currently, DMSO (Di-Methyl-Sulfoxide), human or fetal calf serum (FCS), and glycerol are used as cryoprotective medium to protect the cells against freezing temperatures and water crystal damage, and thus minimize cell mortality. Unfortunately, none of these cryopreservants is capable of preserving the cells over a long period of time, i.e. a few months to a few years. Normally, 30-40% cells die within a few days and over 50% within a few months of live cell cryopreservation (Bravo et al. Burns 26(4), 367-78, 2000), due particularly to cellular damage caused by the formation of water crystals.
There is therefore an urgent need to find a cryopreservant which is capable of forming a thin film around the cells or the tissue and protect them from external aggressions as well as from the formation of water crystals in order to enhance cell viability.